Protracted GLP-1 compounds

ABSTRACT

Novel protracted GLP-1 compounds and therapeutic uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national stage application ofInternational Patent Application PCT/EP2005/055101 (published as WO2006/037810), filed Oct. 7, 2005, which claimed priority of DanishPatent Application PA 2004 01535, filed Oct. 7, 2004; and EuropeanPatent Application 05102168.1, filed Mar. 18, 2005; this applicationfurther claims priority under 35 U.S.C. §119 of U.S. ProvisionalApplication 60/621,022, filed Oct. 21,2004; and U.S. ProvisionalApplication 60/664,498, filed Mar. 23, 2005.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic peptides, i.e.to new protracted GLP-1 compounds.

BACKGROUND OF THE INVENTION

A range of different approaches have been used for modifying thestructure of glucagon-like peptide 1 (GLP-1) compounds in order toprovide a longer duration of action in vivo. WO 96/29342 disclosespeptide hormone derivatives wherein the parent peptide hormone has beenmodified by introducing a lipophilic substituent in the C-terminal aminoacid residue or in the N-terminal amino acid residue.

WO 98/08871 discloses GLP-1 derivatives wherein at least one amino acidresidue of the parent peptide has a lipophilic substituent attached.

WO 99/43708 discloses GLP-1 (7-35) and GLP-1 (7-36) derivatives whichhave a lipophilic substituent attached to the C-terminal amino acidresidue.

WO 00/34331 discloses acylated GLP-1 analogs.

WO 00/69911 discloses activated insulinotropic peptides to be injectedinto patients where they are supposed to react with blood components toform conjugates and thereby allegedly providing longer duration ofaction in vivo.

WO 02/46227 discloses GLP-1 and exendin-4 analogs fused to human serumalbumin in order to extend in vivo half-life.

Many diabetes patients particularly in the type 2 diabetes segment aresubject to so-called “needle-phobia”, i.e. a substantial fear ofinjecting themselves. In the type 2 diabetes segment most patients aretreated with oral hypoglycemic agents, and since GLP-1 compounds areexpected to be the first injectable product these patients will beadministered, the fear of injections may become a serious obstacle forthe widespread use of the clinically very promising GLP-1 compounds.Thus, there is a need to develop new GLP-1 compounds which can beadministered less than once daily, e.g. once every second or third daypreferably once weekly, while retaining an acceptable clinical profile.

SUMMARY OF THE INVENTION

The present invention provides DPP-IV stabilised GLP-1 analogs acylatedwith a diacid. The present invention also provides an acylated GLP-1analog where said GLP-1 analog is stabilised against DPP-IV bymodification of at least one amino acid residue in positions 7 and 8relative to the sequence GLP-1 (7-37) (SEQ ID No 1), and where saidacylation is a diacid attached directly to the C-terminal amino acidresidue of said GLP-1 analog. The present invention also provides aDPP-IV stabilised GLP-1 analog, wherein said GLP-1 analog comprises amodification at the N-terminal, such as a modification of the naturalHis⁷ or Ala⁸.

The present invention also provides a DPP-IV stabilised GLP-1 analogacylated with a diacid, wherein said diacid is attached to the epsilonamino group of a lysine residue of said GLP-1 analog.

The present invention also provides a DPP-IV stabilised GLP-1 analogacylated with a diacid, wherein acylation is on Lys³⁸ of said GLP-1analog.

The present invention also provides a method for increasing the time ofaction in a patient of a GLP-1 analog to more than about 40 hours,characterised in modifying at least one of the amino acid residues inpositions 7 and 8 of a GLP-1 (7-37) peptide or an analog thereof, anddirectly acylating the C-terminal amino acid residue of the GLP-1 analogwith a diacid.

The present invention also provides pharmaceutical compositionscomprising a compound according to the present invention and the use ofcompounds according to the present invention for preparing medicamentsfor treating disease.

DEFINITIONS

In the present specification, the following terms have the indicatedmeaning:

The term “polypeptide” and “peptide” as used herein means a compoundcomposed of at least five constituent amino acids connected by peptidebonds. The constituent amino acids may be from the group of the aminoacids encoded by the genetic code and they may be natural amino acidswhich are not encoded by the genetic code, as well as synthetic aminoacids. Natural amino acids which are not encoded by the genetic code aree.g. hydroxyproline, γ-carboxyglutamate, ornithine, phosphoserine,D-alanine and D-glutamine. Synthetic amino acids comprise amino acidsmanufactured by chemical synthesis, i.e. D-isomers of the amino acidsencoded by the genetic code such as D-alanine and D-leucine, Aib(α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tle(tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid, anthranilicacid.

The term “analog” as used herein referring to a polypeptide means amodified peptide wherein one or more amino acid residues of saidpolypeptide have been substituted by other amino acid residues and/orwherein one or more amino acid residues have been deleted from saidpolypeptide and/or wherein one or more amino acid residues have beendeleted from said polypeptide and or wherein one or more amino acidresidues have been added to the peptide. Such addition or deletion ofamino acid residues can take place at the N-terminal of the peptideand/or at the C-terminal of the peptide.

The term “derivative” as used herein in relation to a peptide means achemically modified peptide or an analogue thereof, wherein at least onesubstituent is not present in the unmodified peptide or an analoguethereof, i.e. a peptide which has been covalently modified. Typicalmodifications are amides, carbohydrates, alkyl groups, acyl groups,esters and the like. An example of a derivative of GLP-1 (7-37) isArg³⁴, Lys²⁶(N^(ε)-(γ-Glu(N^(α)-hexadecanoyl)))-GLP-1(7-37).

The term “GLP-1” as used herein means GLP-1 (7-37) (SEQ ID No:1), ananalog of GLP-1 (7-37), a GLP-1 (7-37) derivative, a derivative of aGLP-1 (7-37) analog or fusion proteins comprising GLP-1 (7-37) or aGLP-1 (7-37) analog or derivative. In one embodiment GLP-1 is aninsulinotropic agent.

The term “insulinotropic agent” as used herein means a compound which isan agonist of the human GLP-1 receptor, i.e. a compound which stimulatesthe formation of cAMP in a suitable medium containing the human GLP-1receptor. The potency of an insulinotropic agent is determined bycalculating the EC₅₀ value from the dose-response curve as describedbelow.

Purified plasma membranes from a stable transfected cell line,BHK467-12A (tk-ts13), expressing the human GLP-1 receptor werestimulated with GLP-1 and peptide analogues, and the potency of cAMPproduction was measured using the AlphaScreen™ cAMP Assay Kit fromPerkin Elmer Life Sciences.

A stable transfected cell line was prepared and a high expressing clonewas selected for screening. The cells were grown at 5% CO₂ in DMEM, 5%FCS, 1% Pen/Strep and 0.5 mg/ml G418.

Cells at approximate 80% confluence were washed twice with PBS andharvested with Versene, centrifuged 5 min at 1000 rpm and thesupernatant removed. The additional steps were all made on ice. The cellpellet was homogenized by the Ultrathurax for 20-30 sec. in 10 ml ofBuffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at20.000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mMNa-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenized for 20-30sec and centrifuged 15 min at 20.000 rpm. Suspension in Buffer 2,homogenization and centrifugation was repeated once and the membraneswere resuspended in Buffer 2 and ready for further analysis or stored at−80° C.

The functional receptor assay was carried out by measuring the peptideinduced cAMP production by The AlphaScreen Technology. The basicprinciple of The AlphaScreen Technology is a competition betweenendogenous cAMP and exogenously added biotin-cAMP. The capture of cAMPis achieved by using a specific antibody conjugated to acceptor beads.Formed cAMP was counted and measured at a AlphaFusion MicroplateAnalyzer. The EC₅₀ values were calculated using the Graph-Pad Prismesoftware.

The term “DPP-IV protected” as used herein in reference to a polypeptidemeans a polypeptide which has been chemically modified in order torender said compound more resistant to the plasma peptidase dipeptidylaminopeptidase-4 (DPP-IV) than the chemically unmodified peptide. TheDPP-IV enzyme in plasma is known to be involved in the degradation ofseveral peptide hormones, e.g. GLP-1, GLP-2, etc.

Resistance of a peptide to degradation by dipeptidyl aminopeptidase IVmay be determined by the following degradation assay:

Aliquots of the peptide are incubated at 37° C. with an aliquot ofpurified dipeptidyl aminopeptidase IV for 4-22 hours in an appropriatebuffer at pH 7-8 (buffer not being albumin). Enzymatic reactions areterminated by the addition of trifluoroacetic acid, and the peptidedegradation products are separated and quantified using HPLC or LC-MSanalysis. One method for performing this analysis is: The mixtures areapplied onto a Zorbax 300SB-C18 (30 nm pores, 5 μm particles) 150×2.1 mmcolumn and eluted at a flow rate of 0.5 ml/min with a linear gradient ofacetonitrile in 0.1% trifluoroacetic acid (0%-100% acetonitrile over 30min). Peptides and their degradation products may be monitored by theirabsorbance at 214 nm (peptide bonds) or 280 nm (aromatic amino acids),and are quantified by integration of their peak areas. The degradationpattern can be determined by using LC-MS where MS spectra of theseparated peak can be determined. Percentage intact/degraded peptide ata given time is used for estimation of the peptide's DPPIV stability.

In one embodiment, a peptide is defined as DPP IV stabilised when it is10 times more stable than the natural peptide based on percentage intactcompound at a given time. Thus, a DPPIV stabilised GLP-1 compound is atleast 10 times more stable than GLP-1 (7-37).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the present invention relates to a DPP-IV stabilised GLP-1analog acylated with a diacid.

In another aspect the present invention relates to an acylated GLP-1analog where said GLP-1 analog is stabilised against DPP-IV bymodification of at least one amino acid residue in positions 7 and 8relative to the sequence GLP-1 (7-37) (SEQ ID No 1), and where saidacylation is a diacid attached directly to the C-terminal amino acidresidue of said GLP-1 analog.

In one embodiment of the invention said diacid is attached to theepsilon amino group of a lysine residue of said GLP-1 analog.

In another embodiment of the invention said GLP-1 analog has an extendedC-terminal, eg. the analog comprises an amino acid residue in position38 relative to the sequence GLP-1 (7-37) (SEQ ID No. 1). Such analogsmay even comprise amino acid residues in position 39, 40, 41 etc.

In another embodiment of the invention said diacid is attached to aLys³⁸ of said GLP-1 analog.

In another embodiment of the invention said diacid is attached to aLys³⁹ of said GLP-1 analog.

In another embodiment of the invention said diacid is attached to aLys⁴⁰ of said GLP-1 analog.

In another embodiment of the invention said diacid is attached to aLys⁴¹ of said GLP-1 analog.

In another embodiment of the invention said diacid is attached to aLys³⁷ of said GLP-1 analog.

In another embodiment of the invention said GLP-1 analog comprises amodification at the N-terminal, such as a modification of the naturalHis⁷ or Ala⁸.

In yet another embodiment of the invention said GLP-1 analog is aposition 7 analog wherein the natural His⁷ has been substituted byimidazopropionyl⁷, α-hydroxy-histidine⁷ or N-methyl-histidine.

In yet another embodiment of the invention said GLP-1 analog is aposition 7 analog wherein the natural His⁷ has been substituted byD-histidine⁷, desamino-histidine⁷, 2-amino-histidine⁷,β-hydroxy-histidine⁷, homohistidine⁷, N^(α)-acetyl-histidine⁷,α-fluoromethyl-histidine⁷, α-methyl-histidine⁷, 3-pyridylalanine⁷,2-pyridylalanine⁷ or 4-pyridylalanine⁷.

In yet another embodiment of the invention said GLP-1 analog is aposition 8 analog wherein the natural Ala⁸ has been substituted by Aib⁸,Gly⁸, Val⁸, Ile⁸, Leu⁸, Ser⁸ or Thr⁸.

In yet another embodiment of the invention said GLP-1 analog comprises asubstitution of the L-alanine in position 8 of GLP-1 (7-37) sequence for(1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid,(1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid,(1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl)carboxylicacid.

In yet another embodiment of the invention said GLP-1 analog comprises asubstitution of the natural Gly in position 22 for a Glu residue.

In yet another embodiment of the invention said GLP-1 analog comprises asubstitution of the natural Val in position 16 for a Leu residue.

In yet another embodiment of the invention said GLP-1 analog comprises asubstitution of the natural Ala in position 30 for a Glu residue.

In yet another embodiment of the invention the diacid is a dicarboxylicacid.

In yet another embodiment of the invention the acylation group is astraight-chain or branched alkane α,ω-dicarboxylic acid.

In yet another embodiment of the invention the acylation group has thestructure HOOC—(CH₂)_(n)CO—, wherein n is 12 to 20.

In yet another embodiment of the invention the acylation group has astructure selected from HOOC—(CH₂)₁₄CO—, HOOC—(CH₂)₁₅CO—,HOOC—(CH₂)₁₆CO—, HOOC—(CH₂)₁₇CO—, and HOOC—(CH₂)₁₈CO—.

In yet another embodiment of the invention the acylation group has thestructure HOOC—(CH₂)₁₆CO—.

In yet another embodiment of the invention said GLP-1 analog comprisesno more than fifteen amino acid residues which have been exchanged,added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1), or no morethan ten amino acid residues which have been exchanged, added or deletedas compared to GLP-1 (7-37) (SEQ ID No. 1).

In yet another embodiment of the invention said GLP-1 analog comprisesno more than six amino acid residues which have been exchanged, added ordeleted as compared to GLP-1 (7-37) (SEQ ID No. 1).

In yet another embodiment of the invention said GLP-1 analog comprisesno more than 6 amino acid residues which are not encoded by the geneticcode.

In yet another embodiment of the invention said GLP-1 analog comprisesno more than 5 amino acid residues which are not encoded by the geneticcode.

In yet another embodiment of the invention said GLP-1 analog comprisesno more than 4 amino acid residues which are not encoded by the geneticcode.

In yet another embodiment of the invention said GLP-1 analog comprisesno more than 3 amino acid residues which are not encoded by the geneticcode.

In yet another embodiment of the invention said GLP-1 analog comprisesno more than 2 amino acid residues which are not encoded by the geneticcode.

In yet another embodiment of the invention said GLP-1 analog comprisesno more than one amino acid residue which is not encoded by the geneticcode.

In yet another embodiment of the invention said GLP-1 analog comprisesonly one lysine residue.

In yet another embodiment of the invention, the DPP-IV stabilised GLP-1analog which is acylated with a diacid directly on the C-terminal aminoacid residue is [Aib8,Arg26,34]GLP-1(7-37)Lys{17-carboxyheptadecanoyl}-OH

or [Gly8,Arg26,34,36]GLP-1 (7-37) Lys(17-Carboxyheptadecanoylamino)-OH

or [AlfahydroxydesaminoHis7, Gly8, Arg26,34]GLP-1(7-37)Lys(17-carboxyheptadecanoyl)-OH

or [Gly8, Glu22,23,30, Arg 18,26,34]GLP1 (7-37)Lys(17-carboxyheptadecanoyl)-NH2

or [Gly8,Arg26,34]GLP-1 (7-37)Lys(19-Carboxynonadecanoyl)-OH

In another aspect the present invention relates to a method forincreasing the time of action in a patient of a GLP-1 analog,characterised in acylating said GLP-1 analog with a diacid directly onthe C-terminal amino acid residue of said GLP-1 analog.

In another aspect the present invention relates to a method forincreasing the time of action in a patient of a GLP-1 analog to morethan about 40 hours, characterised in modifying at least one of theamino acid residues in positions 7 and 8 of a GLP-1 (7-37) peptide or ananalog thereof, and directly acylating the C-terminal amino acid residueof the GLP-1 analog with a diacid.

The GLP-1 analogs can be produced by classical peptide synthesis, e.g.solid phase peptide synthesis using t-Boc or F-Moc chemistry or otherwell established techniques, see the examples and e.g. Houben-Weyl,Methods of organic Chemistry, Volume E 22a, E 22b and E 22 c; Green andWuts, “Protecting Groups in Organic Synthesis”, Jogn Wiley & Sons, 1999.

The therapeutic polypeptide can be produced by a method which comprisesculturing a host cell containing a DNA sequence encoding the polypeptideand capable of expressing the polypeptide in a suitable nutrient mediumunder conditions permitting the expression of the peptide, after whichthe resulting peptide is recovered from the culture.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of peptide in question.

The DNA sequence encoding the therapeutic polypeptide may suitably be ofgenomic or cDNA origin, for instance obtained by preparing a genomic orcDNA library and screening for DNA sequences coding for all or part ofthe polypeptide by hybridisation using synthetic oligonucleotide probesin accordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the polypeptide may also be prepared synthetically byestablished standard methods, e.g. the phosphoramidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.The DNA sequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the peptide is operably linked to additional segments requiredfor transcription of the DNA, such as a promoter. The promoter may beany DNA sequence which shows transcriptional activity in the host cellof choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding thepeptide of the invention in a variety of host cells are well known inthe art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the peptide may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a parent peptide of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the peptide in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe peptide. The secretory signal sequence may be that normallyassociated with the peptide or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the presentpeptide, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the presentpeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.Examples of suitable host cells well known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

Examples of compounds which can be useful as GLP-1 moieties according tothe present invention are described in International Patent ApplicationNo. WO 87/06941 (The General Hospital Corporation) which relates to apeptide fragment which comprises GLP-1 (7-37) and functional derivativesthereof and to its use as an insulinotropic agent.

Further GLP-1 analogues are described in International PatentApplication No. 90/11296 (The General Hospital Corporation) whichrelates to peptide fragments which comprise GLP-1 (7-36) and functionalderivatives thereof and have an insulinotropic activity which exceedsthe insulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37) and to theiruse as insulinotropic agents.

International Patent Application No. 91/11457 (Buckley et al.) disclosesanalogues of the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 whichcan also be useful as GLP-1 moieties according to the present invention.

Pharmaceutical Compositions

Pharmaceutical compositions containing a compound according to thepresent invention may be prepared by conventional techniques, e.g. asdescribed in Remington's Pharmaceutical Sciences, 1985 or in Remington:The Science and Practice of Pharmacy, 19^(th) edition, 1995.

One object of the present invention is to provide a pharmaceuticalformulation comprising a compound according to the present inventionwhich is present in a concentration from about 0.1 mg/ml to about 25mg/ml, and wherein said formulation has a pH from 2.0 to 10.0. Thepharmaceutical formulation may comprise a compound according to thepresent invention which is present in a concentration from about 0.1mg/ml to about 50 mg/ml, and wherein said formulation has a pH from 2.0to 10.0. The formulation may further comprise a buffer system,preservative(s), isotonicity agent(s), chelating agent(s), stabilizersand surfactants. In one embodiment of the invention the pharmaceuticalformulation is an aqueous formulation, i.e. formulation comprisingwater. Such formulation is typically a solution or a suspension. In afurther embodiment of the invention the pharmaceutical formulation is anaqueous solution. The term “aqueous formulation” is defined as aformulation comprising at least 50% w/w water. Likewise, the term“aqueous solution” is defined as a solution comprising at least 50% w/wwater, and the term “aqueous suspension” is defined as a suspensioncomprising at least 50% w/w water.

In another embodiment the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect the invention relates to a pharmaceuticalformulation comprising an aqueous solution of a compound according tothe present invention, and a buffer, wherein said compound is present ina concentration from 0.1 mg/ml or above, and wherein said formulationhas a pH from about 2.0 to about 10.0.

In a further aspect the invention relates to a pharmaceuticalformulation comprising an aqueous solution of a compound according tothe present invention, and a buffer, wherein said compound is present ina concentration from 0.1 mg/ml or above, and wherein said formulationhas a pH from about 7.0 to about 8.5.

In a another embodiment of the invention the pH of the formulation isselected from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,9.7, 9.8, 9.9, and 10.0. Preferably, the pH of the formulation is atleast 1 pH unit from the isoelectric point of the compound according tothe present invention, even more preferable the pH of the formulation isat least 2 pH unit from the isoelectric point of the compound accordingto the present invention.

In a further embodiment of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethane, hepes, bicine, tricine, malic acid,succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid ormixtures thereof. Each one of these specific buffers constitutes analternative embodiment of the invention.

In a further embodiment of the invention the formulation furthercomprises a pharmaceutically acceptable preservative. In a furtherembodiment of the invention the preservative is selected from the groupconsisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, ethanol,chlorobutanol, and thimerosal, bronopol, benzoic acid, imidurea,chlorohexidine, sodium dehydroacetate, chlorocresol, ethylp-hydroxybenzoate, benzethonium chloride, chlorphenesine(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a furtherembodiment of the invention the preservative is present in aconcentration from 0.1 mg/ml to 30 mg/ml. In a further embodiment of theinvention the preservative is present in a concentration from 0.1 mg/mlto 20 mg/ml. In a further embodiment of the invention the preservativeis present in a concentration from 0.1 mg/ml to 5 mg/ml. In a furtherembodiment of the invention the preservative is present in aconcentration from 5 mg/ml to 10 mg/ml. In a further embodiment of theinvention the preservative is present in a concentration from 10 mg/mlto 20 mg/ml. Each one of these specific preservatives constitutes analternative embodiment of the invention. The use of a preservative inpharmaceutical compositions is well-known to the skilled person. Forconvenience reference is made to Remington: The Science and Practice ofPharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises an isotonic agent. In a further embodiment of the inventionthe isotonic agent is selected from the group consisting of a salt (e.g.sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such asmono-, di-, or polysaccharides, or water-soluble glucans, including forexample fructose, glucose, mannose, sorbose, xylose, maltose, lactose,sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, solublestarch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.In one embodiment the sugar additive is sucrose. Sugar alcohol isdefined as a C4-C8 hydrocarbon having at least one —OH group andincludes, for example, mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. In one embodiment the sugar alcoholadditive is mannitol. The sugars or sugar alcohols mentioned above maybe used individually or in combination. There is no fixed limit to theamount used, as long as the sugar or sugar alcohol is soluble in theliquid preparation and does not adversely effect the stabilizing effectsachieved using the methods of the invention. In one embodiment, thesugar or sugar alcohol concentration is between about 1 mg/ml and about150 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 1 mg/ml to 50 mg/ml. In a furtherembodiment of the invention the isotonic agent is present in aconcentration from 1 mg/ml to 7 mg/ml. In a further embodiment of theinvention the isotonic agent is present in a concentration from 8 mg/mlto 24 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 25 mg/ml to 50 mg/ml. Each one ofthese specific isotonic agents constitutes an alternative embodiment ofthe invention. The use of an isotonic agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises a chelating agent. In a further embodiment of the inventionthe chelating agent is selected from salts of ethylenediaminetetraaceticacid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In afurther embodiment of the invention the chelating agent is present in aconcentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of theinvention the chelating agent is present in a concentration from 0.1mg/ml to 2 mg/ml. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/ml to 5 mg/ml. Each one ofthese specific chelating agents constitutes an alternative embodiment ofthe invention. The use of a chelating agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises a stabiliser. The use of a stabilizer in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a polypeptide that possibly exhibits aggregate formation duringstorage in liquid pharmaceutical formulations. By “aggregate formation”is intended a physical interaction between the polypeptide moleculesthat results in formation of oligomers, which may remain soluble, orlarge visible aggregates that precipitate from the solution. By “duringstorage” is intended a liquid pharmaceutical composition or formulationonce prepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or formulation is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli (1984) J.Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) inSpray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez,U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm.18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), orair drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser(1991) Biopharm. 4:47-53). Aggregate formation by a polypeptide duringstorage of a liquid pharmaceutical composition can adversely affectbiological activity of that polypeptide, resulting in loss oftherapeutic efficacy of the pharmaceutical composition. Furthermore,aggregate formation may cause other problems such as blockage of tubing,membranes, or pumps when the polypeptide-containing pharmaceuticalcomposition is administered using an infusion system.

The pharmaceutical compositions of the invention may further comprise anamount of an amino acid base sufficient to decrease aggregate formationby the polypeptide during storage of the composition. By “amino acidbase” is intended an amino acid or a combination of amino acids, whereany given amino acid is present either in its free base form or in itssalt form. Where a combination of amino acids is used, all of the aminoacids may be present in their free base forms, all may be present intheir salt forms, or some may be present in their free base forms whileothers are present in their salt forms. In one embodiment, amino acidsused for preparing the compositions of the invention are those carryinga charged side chain, such as arginine, lysine, aspartic acid, andglutamic acid. In one embodiment, the amino acid used for preparing thecompositions of the invention is glycine. Any stereoisomer (i.e. L or D)of a particular amino acid (e.g. methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine andmixtures thereof) or combinations of these stereoisomers, may be presentin the pharmaceutical compositions of the invention so long as theparticular amino acid is present either in its free base form or itssalt form. In one embodiment the L-stereoisomer is used. Compositions ofthe invention may also be formulated with analogues of these aminoacids. By “amino acid analogue” is intended a derivative of thenaturally occurring amino acid that brings about the desired effect ofdecreasing aggregate formation by the polypeptide during storage of theliquid pharmaceutical compositions of the invention. Suitable arginineanalogues include, for example, aminoguanidine, ornithine andN-monoethyl L-arginine, suitable methionine analogues include ethionineand buthionine and suitable cysteine analogues include S-methyl-Lcysteine. As with the other amino acids, the amino acid analogues areincorporated into the compositions in either their free base form ortheir salt form. In a further embodiment of the invention the aminoacids or amino acid analogues are used in a concentration, which issufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention methionine (or other sulphuricamino acids or amino acid analogous) may be added to inhibit oxidationof methionine residues to methionine sulfoxide when the polypeptideacting as the therapeutic agent is a polypeptide comprising at least onemethionine residue susceptible to such oxidation. By “inhibit” isintended minimal accumulation of methionine oxidized species over time.Inhibiting methionine oxidation results in greater retention of thepolypeptide in its proper molecular form. Any stereoisomer of methionine(L, D or a mixture thereof) can be used. The amount to be added shouldbe an amount sufficient to inhibit oxidation of the methionine residuessuch that the amount of methionine sulfoxide is acceptable to regulatoryagencies. Typically, this means that the composition contains no morethan about 10% to about 30% methionine sulfoxide. Generally, this can beachieved by adding methionine such that the ratio of methionine added tomethionine residues ranges from about 1:1 to about 1000:1, such as 10:1to about 100:1.

In a further embodiment of the invention the formulation furthercomprises a stabiliser selected from the group of high molecular weightpolymers or low molecular compounds. In a further embodiment of theinvention the stabilizer is selected from polyethylene glycol (e.g. PEG3350), polyvinylalcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). Each one of these specificstabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizingagents, which further enhance stability of a therapeutically activepolypeptide therein. Stabilizing agents of particular interest to thepresent invention include, but are not limited to, methionine and EDTA,which protect the polypeptide against methionine oxidation, and anonionic surfactant, which protects the polypeptide against aggregationassociated with freeze-thawing or mechanical shearing.

In a further embodiment of the invention the formulation furthercomprises a surfactant. In a further embodiment of the invention thesurfactant is selected from a detergent, ethoxylated castor oil,polyglycolized glycerides, acetylated monoglycerides, sorbitan fattyacid esters, polyoxypropylene-polyoxyethylene block polymers (eg.poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100),polyoxyethylene sorbitan fatty acid esters, starshaped PEO,polyoxyethylene and polyethylene derivatives such as alkylated andalkoxylated derivatives (tweens, e.g. Tween-20, Tween-40, Tween-80 andBrij-35), polyoxyethylene hydroxystearate, monoglycerides or ethoxylatedderivatives thereof, diglycerides or polyoxyethylene derivativesthereof, alcohols, glycerol, lecitins and phospholipids (eg.phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin),derivates of phospholipids (eg. dipalmitoyl phosphatidic acid) andlysophospholipids (eg. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidyl-threonine, andglycerophospholipids (eg. cephalins), glyceroglycolipids (eg.galacto-pyransoide), sphingoglycolipids (eg. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (eg. oleic acid and caprylic acid),acylcarnitines and derivatives, N^(α)-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, N^(α)-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, N^(α)-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate),sodium caprylate, cholic acid or derivatives thereof, bile acids andsalts thereof and glycine or taurine conjugates, ursodeoxycholic acid,sodium cholate, sodium deoxycholate, sodium taurocholate, sodiumglycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethyl-ammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationicsurfactants (quarternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecylβ-D-glucopyranoside), poloxamines (eg. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof. Each one of these specific surfactants constitutes analternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

A composition for parenteral administration of GLP-1 compounds may, forexample, be prepared as described in WO 03/002136.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatin orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing a compound according to thepresent invention may be administered to a patient in need of suchtreatment at several sites, for example, at topical sites, for example,skin and mucosal sites, at sites which bypass absorption, for example,administration in an artery, in a vein, in the heart, and at sites whichinvolve absorption, for example, administration in the skin, under theskin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, rinses, capsules, for example, hardgelatine capsules and soft gelatine capsules, suppositories, rectalcapsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops,ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginalrings, vaginal ointments, injection solution, in situ transformingsolutions, for example in situ gelling, in situ setting, in situprecipitating, in situ crystallization, infusion solution, and implants.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the compound,increase bioavailability, increase solubility, decrease adverse effects,achieve chronotherapy well known to those skilled in the art, andincrease patient compliance or any combination thereof. Examples ofcarriers, drug delivery systems and advanced drug delivery systemsinclude, but are not limited to, polymers, for example cellulose andderivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,L2 phase and dispersions there of, well known to those skilled in theart of phase behaviour in lipid-water systems, polymeric micelles,multiple emulsions, self-emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

Compositions of the current invention are useful in the formulation ofsolids, semisolids, powder and solutions for pulmonary administration ofthe compound, using, for example a metered dose inhaler, dry powderinhaler and a nebulizer, all being devices well known to those skilledin the art.

Compositions of the current invention are specifically useful in theformulation of controlled, sustained, protracting, retarded, and slowrelease drug delivery systems. More specifically, but not limited to,compositions are useful in formulation of parenteral controlled releaseand sustained release systems (both systems leading to a many-foldreduction in number of administrations), well known to those skilled inthe art. Even more preferably, are controlled release and sustainedrelease systems administered subcutaneous. Without limiting the scope ofthe invention, examples of useful controlled release system andcompositions are hydrogels, oleaginous gels, liquid crystals, polymericmicelles, microspheres, nanoparticles,

Methods to produce controlled release systems useful for compositions ofthe current invention include, but are not limited to, crystallization,condensation, co-crystallization, precipitation, co-precipitation,emulsification, dispersion, high pressure homogenization, encapsulation,spray drying, microencapsulation, coacervation, phase separation,solvent evaporation to produce microspheres, extrusion and supercriticalfluid processes. General reference is made to Handbook of PharmaceuticalControlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) andDrug and the Pharmaceutical Sciences vol. 99: Protein Formulation andDelivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a solution or suspension for theadministration of the compound according to the present invention in theform of a nasal or pulmonal spray. As a still further option, thepharmaceutical compositions containing the compound of the invention canalso be adapted to transdermal administration, e.g. by needle-freeinjection or from a patch, optionally an iontophoretic patch, ortransmucosal, e.g. buccal, administration.

The term “stabilized formulation” refers to a formulation with increasedphysical stability, increased chemical stability or increased physicaland chemical stability.

The term “physical stability” of the protein formulation as used hereinrefers to the tendency of the protein to form biologically inactiveand/or insoluble aggregates of the protein as a result of exposure ofthe protein to thermo-mechanical stresses and/or interaction withinterfaces and surfaces that are destabilizing, such as hydrophobicsurfaces and interfaces. Physical stability of the aqueous proteinformulations is evaluated by means of visual inspection and/or turbiditymeasurements after exposing the formulation filled in suitablecontainers (e.g. cartridges or vials) to mechanical/physical stress(e.g. agitation) at different temperatures for various time periods.Visual inspection of the formulations is performed in a sharp focusedlight with a dark background. The turbidity of the formulation ischaracterized by a visual score ranking the degree of turbidity forinstance on a scale from 0 to 3 (a formulation showing no turbiditycorresponds to a visual score 0, and a formulation showing visualturbidity in daylight corresponds to visual score 3). A formulation isclassified physical unstable with respect to protein aggregation, whenit shows visual turbidity in daylight. Alternatively, the turbidity ofthe formulation can be evaluated by simple turbidity measurementswell-known to the skilled person. Physical stability of the aqueousprotein formulations can also be evaluated by using a spectroscopicagent or probe of the conformational status of the protein. The probe ispreferably a small molecule that preferentially binds to a non-nativeconformer of the protein. One example of a small molecular spectroscopicprobe of protein structure is Thioflavin T. Thioflavin T is afluorescent dye that has been widely used for the detection of amyloidfibrils. In the presence of fibrils, and perhaps other proteinconfigurations as well, Thioflavin T gives rise to a new excitationmaximum at about 450 nm and enhanced emission at about 482 nm when boundto a fibril protein form. Unbound Thioflavin T is essentiallynon-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in proteinstructure from native to non-native states. For instance the“hydrophobic patch” probes that bind preferentially to exposedhydrophobic patches of a protein. The hydrophobic patches are generallyburied within the tertiary structure of a protein in its native state,but become exposed as a protein begins to unfold or denature. Examplesof these small molecular, spectroscopic probes are aromatic, hydrophobicdyes, such as anthracene, acridine, phenanthroline or the like. Otherspectroscopic probes are metal-amino acid complexes, such as cobaltmetal complexes of hydrophobic amino acids, such as phenylalanine,leucine, isoleucine, methionine, and valine, or the like.

The term “chemical stability” of the protein formulation as used hereinrefers to chemical covalent changes in the protein structure leading toformation of chemical degradation products with potential lessbiological potency and/or potential increased immunogenic propertiescompared to the native protein structure. Various chemical degradationproducts can be formed depending on the type and nature of the nativeprotein and the environment to which the protein is exposed. Eliminationof chemical degradation can most probably not be completely avoided andincreasing amounts of chemical degradation products is often seen duringstorage and use of the protein formulation as well-known by the personskilled in the art. Most proteins are prone to deamidation, a process inwhich the side chain amide group in glutaminyl or asparaginyl residuesis hydrolysed to form a free carboxylic acid. Other degradationspathways involves formation of high molecular weight transformationproducts where two or more protein molecules are covalently bound toeach other through transamidation and/or disulfide interactions leadingto formation of covalently bound dimer, oligomer and polymer degradationproducts (Stability of Protein Pharmaceuticals, Ahern. T. J. & ManningM. C., Plenum Press, New York 1992). Oxidation (of for instancemethionine residues) can be mentioned as another variant of chemicaldegradation. The chemical stability of the protein formulation can beevaluated by measuring the amount of the chemical degradation productsat various time-points after exposure to different environmentalconditions (the formation of degradation products can often beaccelerated by for instance increasing temperature). The amount of eachindividual degradation product is often determined by separation of thedegradation products depending on molecule size and/or charge usingvarious chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a “stabilized formulation” refers to aformulation with increased physical stability, increased chemicalstability or increased physical and chemical stability. In general, aformulation must be stable during use and storage (in compliance withrecommended use and storage conditions) until the expiration date isreached.

In one embodiment of the invention the pharmaceutical formulationcomprising the compound according to the present invention is stable formore than 6 weeks of usage and for more than 3 years of storage.

In another embodiment of the invention the pharmaceutical formulationcomprising the compound according to the present invention is stable formore than 4 weeks of usage and for more than 3 years of storage.

In a further embodiment of the invention the pharmaceutical formulationcomprising the compound according to the present invention is stable formore than 4 weeks of usage and for more than two years of storage.

In an even further embodiment of the invention the pharmaceuticalformulation comprising the compound is stable for more than 2 weeks ofusage and for more than two years of storage.

Pharmaceutical compositions containing a GLP-1 derivative according tothe present invention may be administered parenterally to patients inneed of such a treatment. Parenteral administration may be performed bysubcutaneous, intramuscular or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a powder or a liquid for theadministration of the GLP-1 derivative in the form of a nasal orpulmonal spray. As a still further option, the GLP-1 derivatives of theinvention can also be administered transdermally, e.g. from a patch,optionally a iontophoretic patch, or transmucosally, e.g. bucally.

Thus, the injectable compositions of the GLP-1 derivative of theinvention can be prepared using the conventional techniques of thepharmaceutical industry which involves dissolving and mixing theingredients as appropriate to give the desired end product.

According to one procedure, the GLP-1 derivative is dissolved in anamount of water which is somewhat less than the final volume of thecomposition to be prepared. An isotonic agent, a preservative and abuffer is added as required and the pH value of the solution isadjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base,e.g. aqueous sodium hydroxide as needed. Finally, the volume of thesolution is adjusted with water to give the desired concentration of theingredients.

Further to the above-mentioned components, solutions containing a GLP-1derivative according to the present invention may also contain asurfactant in order to improve the solubility and/or the stability ofthe GLP-1 derivative.

A composition for nasal administration of certain peptides may, forexample, be prepared as described in European Patent No. 272097 (to NovoNordisk A/S) or in WO 93/18785.

According to one preferred embodiment of the present invention, theGLP-1 derivative is provided in the form of a composition suitable foradministration by injection. Such a composition can either be aninjectable solution ready for use or it can be an amount of a solidcomposition, e.g. a lyophilised product, which has to be dissolved in asolvent before it can be injected. The injectable solution preferablycontains not less than about 2 mg/ml, preferably not less than about 5mg/ml, more preferred not less than about 10 mg/ml of the GLP-1derivative and, preferably, not more than about 100 mg/ml of the GLP-1derivative.

The GLP-1 derivatives of this invention can be used in the treatment ofvarious diseases. The particular GLP-1 derivative to be used and theoptimal dose level for any patient will depend on the disease to betreated and on a variety of factors including the efficacy of thespecific peptide derivative employed, the age, body weight, physicalactivity, and diet of the patient, on a possible combination with otherdrugs, and on the severity of the case. It is recommended that thedosage of the GLP-1 derivative of this invention be determined for eachindividual patient by those skilled in the art.

In particular, it is envisaged that the GLP-1 derivative will be usefulfor the preparation of a medicament with a protracted profile of actionfor the treatment of non-insulin dependent diabetes mellitus and/or forthe treatment of obesity.

In another aspect the present invention relates to the use of a compoundaccording to the invention for the preparation of a medicament.

In one embodiment the present invention relates to the use of a compoundaccording to the invention for the preparation of a medicament for thetreatment of hyperglycemia, type 2 diabetes, impaired glucose tolerance,type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, β-cellapoptosis, β-cell deficiency, myocardial infarction, inflammatory bowelsyndrome, dyspepsia, cognitive disorders, e.g. cognitive enhancing,neuroprotection, atherosclerosis, coronary heart disease and othercardiovascular disorders.

In another embodiment the present invention relates to the use of acompound according to the invention for the preparation of a medicamentfor the treatment of small bowel syndrome, inflammatory bowel syndromeor Crohns disease.

In another embodiment the present invention relates to the use of acompound according to the invention for the preparation of a medicamentfor the treatment of hyperglycemia, type 1 diabetes, type 2 diabetes orβ-cell deficiency.

The treatment with a compound according to the present invention mayalso be combined with combined with a second or more pharmacologicallyactive substances, e.g. selected from antidiabetic agents, antiobesityagents, appetite regulating agents, antihypertensive agents, agents forthe treatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity. In the present context the expression “antidiabetic agent”includes compounds for the treatment and/or prophylaxis of insulinresistance and diseases wherein insulin resistance is thepathophysiological mechanism.

Examples of these pharmacologically active substances are: Insulin,GLP-1 agonists, sulphonylureas (e.g. tolbutamide, glibenclamide,glipizide and gliclazide), biguanides e.g. metformin, meglitinides,glucosidase inhibitors (e.g. acorbose), glucagon antagonists, DPP-IV(dipeptidyl peptidase-IV) inhibitors, gastrin, inhibitors of hepaticenzymes involved in stimulation of gluconeogenesis and/orglycogenolysis, glucose uptake modulators, thiazolidinediones such astroglitazone and ciglitazone, compounds modifying the lipid metabolismsuch as antihyperlipidemic agents as HMG CoA inhibitors (statins),compounds lowering food intake, RXR agonists and agents acting on theATP-dependent potassium channel of the β-cells, e.g. glibenclamide,glipizide, gliclazide and repaglinide; Cholestyramine, colestipol,clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol,dextrothyroxine, nateglinide, repaglinide; β-blockers such asalprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE(angiotensin converting enzyme) inhibitors such as benazepril,captopril, enalapril, fosinopril, lisinopril, alatriopril, quinapril andramipril, calcium channel blockers such as nifedipine, felodipine,nicardipine, isradipine, nimodipine, diltiazem and verapamil, andα-blockers such as doxazocin, urapidil, prazosin and terazosin; CART(cocaine amphetamine regulated transcript) agonists, NPY (neuropeptideY) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF(tumor necrosis factor) agonists, CRF (corticotropin releasing factor)agonists, CRF BP (corticotropin releasing factor binding protein)antagonists, urocortin agonists, β3 agonists, MSH(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentratinghormone) antagonists, CCK (cholecystokinin) agonists, serotoninre-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors,mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth hormonereleasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DAagonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR(retinoid X receptor) modulators, TR β agonists; histamine H3antagonists, gastrin and gastrin analogs.

It should be understood that any suitable combination of the compoundsaccording to the invention with one or more of the above-mentionedcompounds and optionally one or more further pharmacologically activesubstances are considered to be within the scope of the presentinvention.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES

Abbreviations Used:

r.t Room temperature

DIEA diisopropylethylamine

H₂O water

CH₃CN acetonitrile

DMF NN dimethylformamide

HBTU 2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate

Fmoc 9 H-fluoren-9-ylmethoxycarbonyl

Boc tert butyloxycarbonyl

OtBu tert butyl ester

tBu tert butyl

Trt triphenylmethyl

Pmc 2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl

Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl

DCM dichloromethane

TIS triisopropylsilane)

TFA: trifluoroacetic acid

Et₂O: diethylether

NMP 1-Methyl-pyrrolidin-2-one

The peptide was synthesized on Fmoc protected Rink amide resin(Novabiochem) or chlorotrityl resin using Fmoc strategy on an AppliedBiosystems 433A peptide synthesizer in 0.25 mmol scale using themanufacturer supplied FastMoc UV protocols which employ HBTU(2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate) mediated couplings in N-methylpyrrolidone(N-methylpyrrolidone) and UV monitoring of the deprotection of the Fmocprotection group. The protected amino acid derivatives used werestandard Fmoc-amino acids (Anaspec) supplied in preweighed cartridgessuitable for the ABI433A synthesizer with the exception of unnaturalaminoacids such as Fmoc-Aib-OH (Fmoc-aminoisobutyric acid).

The attachment of sidechains and linkers to specific lysine residues onthe crude resin bound protected peptide was carried out in a specificposition by incorporation of Fmoc-Lys(Dde)-OH during automated synthesisfollowed by selective deprotection with hydrazine.

Procedure for removal of Dde-protection. The resin (0.25 mmol) wasplaced in a manual shaker/filtration apparatus and treated with 2%hydrazine in N-methylpyrrolidone (20 ml, 2×12 min) to remove the DDEgroup and wash with N-methylpyrrolidone (4×20 ml).

Procedure for Attachment of Sidechains to Lysine Residues.

The amino acid (4 molar equivalents relative to resin) was dissolved inN-methyl pyrrolidone/methylene chloride (1:1, 10 ml).Hydroxybenzotriazole (HOBt) (4 molar equivalents relative to resin) anddiisopropylcarbodiimide (4 molar equivalents relative to resin) wasadded and the solution was stirred for 15 min. The solution was added tothe resin and diisopropyethylamine (4 molar equivalents relative toresin) was added. The resin was shaken 24 hours at room temperature. Theresin was washed with N-methylpyrrolidone (2×20 ml),N-methylpyrrolidone/Methylene chloride (1:1) (2×20 ml) and methylenechloride (2×20 ml).

Procedure for removal of Fmoc-protection: The resin (0.25 mmol) wasplaced in a filter flask in a manual shaking apparatus and treated withN-methylpyrrolidone/methylene chloride (1:1) (2×20 ml) and withN-methylpyrrolidone (1×20 ml), a solution of 20% piperidine inN-methylpyrrolidone (3×20 ml, 10 min each). The resin was washed withN-methyl pyrrolidone (2×20 ml), N-methylpyrrolidone/Methylene chloride(1:1) (2×20 ml) and methylene chloride (2×20 ml).

Procedure for Cleaving the Peptide Off the Resin:

The peptide was cleaved from the resin by stirring for 180 min at roomtemperature with a mixture of trifluoroacetic acid, water andtriisopropylsilane (95:2.5:2.5). The cleavage mixture was filtered andthe filtrate was concentrated to an oil by a stream of nitrogen. Thecrude peptide was precipitated from this oil with 45 ml diethyl etherand washed 3 times with 45 ml diethyl ether.

Purification: The crude peptide was purified by semipreparative HPLC ona 20 mm×250 mm column packed with 7μ C-18 silica. Depending on thepeptide two one or two purification systems were used.

TFA: After drying the crude peptide was dissolved in 5 ml 50% aceticacid H₂O and diluted to 20 ml with H₂O and injected on the column whichthen was eluted with a gradient of 40-60% CH₃CN in 0.1% TFA 10 ml/minduring 50 min at 40° C. The peptide containing fractions were collected.The purified peptide was lyophilized after dilution of the eluate withwater.

Ammonium sulphate: The column was equilibrated with 40% CH₃CN in 0.05M(NH₄)₂SO₄, which was adjusted to pH 2.5 with concentrated H₂SO₄. Afterdrying the crude peptide was dissolved in 5 ml 50% acetic acid H₂O anddiluted to 20 ml with H₂O and injected on the column which then waseluted with a gradient of 40%-60% CH₃CN in 0.05M (NH₄)₂SO₄, pH 2.5 at 10ml/min during 50 min at 40° C. The peptide containing fractions werecollected and diluted with 3 volumes of H₂O and passed through aSep-Pak® C18 cartridge (Waters part. #:51910) which has beenequilibrated with 0.1% TFA. It was then eluted with 70% CH₃CN containing0.1% TFA and the purified peptide was isolated by lyophilisation afterdilution of the eluate with water.

The final product obtained was characterised by analytical RP-HPLC(retention time) and by LCMS

The RP-HPLC analysis was performed using UV detection at 214 nm and aVydac 218TP54 4.6 mm×250 mm 5μ C-18 silica column (The SeparationsGroup, Hesperia, USA) which was eluted at 1 ml/min at 42° C. Twodifferent elution conditions were used:

-   A1: Equilibration of the column with in a buffer consisting of 0.1 M    (NH₄)₂SO₄, which was adjusted to pH 2.5 with concentrated H₂SO₄ and    elution by a gradient of 0% to 60% CH₃CN in the same buffer during    50 min.-   B1: Equilibration of the column with 0.1% TFA/H₂O and elution by a    gradient of 0% CH₃CN/0.1% TFA/H₂O to 60% CH₃CN/0.1% TFA/H₂O during    50 min.-   B6: Equilibration of the column with 0.1% TFA/H₂O and elution by a    gradient of 0% CH₃CN/0.1% TFA/H₂O to 90% CH₃CN/0.1% TFA/H₂O during    50 min.

LCMS was performed on a setup consisting of Hewlett Packard series 1100G1312A Bin Pump, Hewlett Packard series 1100 Column compartment, HewlettPackard series 1100 G1315A DAD diode array detector, Hewlett Packardseries 1100 MSD and Sedere 75 Evaporative Light Scatteringdetectorcontrolled by HP Chemstation software. The HPLC pump isconnected to two eluent reservoirs containing:

A: 10 mM NH₄OH in water

B: 10 mM NH₄OH in 90% acetonitrile

The analysis was performed at 23° C. by injecting an appropriate volumeof the sample (preferably 20 μl) onto the column which is eluted with agradient of A and B.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following table.

-   Column Waters Xterra MS C-18×3 mm id 5 μm-   Gradient 5%-100% acetonitrile linear during 6.5 min at 1.5 ml/min-   Detection 210 nm (analogue output from DAD)-   ELS (analogue output from ELS)-   MS ionisation mode API-ES. Scan 100-1000 amu step 0.1 amu

Example 1 [Aib8,Arg26,34]GLP-1 (7-37)Lys{17-carboxyheptadecanoyl}-OH

A resin (Rink amide, 0.68 mmol/g Novabiochem 0.25 mmole) was used toproduce the primary sequence on an ABI433A machine according tomanufacturers guidelines. All protecting groups were acid labile withthe exception of the residue used in position 37 (FmocLys(ivDde)-OH,Novabiochem) allowing specific deprotection of this lysine rather thanany other lysine.

Procedure

The resin (0.25 mmole) was placed in a manual shaker/filtrationapparatus and treated with 2% hydrazine in N-methylpyrrolidone in (2×12min. 2×20 ml) to remove the Dde group. The resin was washed withN-methylpyrrolidone (4×20 ml). Fmoc-8-amino-3,6-dioxaoctanoic acid(Neosystem FA03202) (4 molar equivalents relative to resin) wasdissolved in N-methyl pyrrolidone/methylene chloride (1:1, 20 ml).Hydroxybenzotriazole (HOBt) (4 molar equivalents relative to resin) anddiisopropylcarbodiimide (4 molar equivalents relative to resin) wasadded and the solution was stirred for 15 min. The solution was added tothe resin and diisopropylethylamine (4 molar equivalents relative toresin) was added. The resin was shaken 24 hours at room temperature. Theresin was washed with N-methylpyrrolidone (4×20 ml). A solution of 20%piperidine in N-methylpyrrolidone (3×20 ml, 10 min each) was added tothe resin while shaking. The resin was washed with N-methylpyrrolidone(4×20 ml). Dodecanoic acid (4 molar equivalents relative to resin) wasdissolved in N-methyl pyrrolidone/methylene chloride (1:1, 20 ml).Hydroxybenzotriazole hydrate (HOBt; H₂O) (4 molar equivalents relativeto resin) and diisopropylcarbodiimide (4 molar equivalents relative toresin) were added and the solution was stirred for 15 min. The solutionwas added to the resin and diisopropylethylamine (4 molar equivalentsrelative to resin) was added. The resin was shaken 24 hours at roomtemperature. The resin was washed with N-methylpyrrolidone (2×20 ml),N-methylpyrrolidone/methylene chloride (1:1) (2×20 ml) and methylenechloride (2×20 ml). The peptide was cleaved from the resin by stirringfor 180 min at room temperature with a mixture of trifluoroacetic acid,water and triisopropylsilane (95:2.5:2.5 15 ml). The cleavage mixturewas filtered and the filtrate was concentrated to an oil in vacuum. Thecrude peptide was precipitated from this oil with 45 ml diethyl etherand washed 3 times with 45 ml diethyl ether. The crude peptide waspurified by preparative HPLC on a 20 mm×250 mm column packed with 7μC-18 silica. The crude peptide was dissolved in 5 ml 50% acetic acid inwater and diluted to 20 ml with H₂O and injected on the column whichthen was eluted with a gradient of 40-60% (CH₃CN in water with 0.1% TFA)10 ml/min during 50 min at 40° C. The peptide containing fractions werecollected. The purified peptide was lyophilized after dilution of theeluate with water.

HPLC (method A1): RT=43.82 min

LCMS: m/z=1284.8 (M+3H)³⁺ Calculated (M+H)⁺=3850.8

Example 2 [Gly8,Arg26,34,36]GLP-1 (7-37)Lys(17-Carboxyheptadecanoylamino)-OH

Prepared as in Example 1 and in accordance with “Synthetic methods”.

HPLC (method A1): RT=43.6 min

LCMS: m/z=1275.6 (M+3H)³⁺ Calculated (M+H)⁺=3822.1

Example 3 [AlfahydroxydesaminoHis7, Gly8, Arg26,34]GLP-1(7-37)Lys(17-carboxyheptadecanoyl)-OH

Prepared similar to Example 1 and in accordance with “Syntheticmethods”.

HPLC (method A1): RT=33.6 min

LCMS: m/z=1275.7 (M+3H)³⁺ Calculated (M+H)⁺=3823.3

Example 4 [Gly8, Glu22,23,30, Arg 18,26,34]GLP1 (7-37)Lys(17-carboxyheptadecanoyl)-NH₂

Prepared similar to Example 1 and in accordance with “Syntheticmethods”.

LCMS: m/z=1342.1 (M+3H)³⁺ Calculated (M+H)⁺=4021.6

Example 5 [Gly8,Arg26,34]GLP-1 (7-37)Lys(19-Carboxynonadecanoyl)-OH

Prepared similar as Example 1 and in accordance with “Syntheticmethods”.

HPLC (method A1): RT=46.6 min

LCMS: m/z=1284.7 (M+3H)³⁺ Calculated (M+H)⁺=3850.4

1. An acylated GLP-1 analog where said GLP-1 analog has a sequenceaccording to SEQ ID NO: 1 7 8 9 10 11 12 13 14 15 16 17His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser- 18 19 20 21 22 23 24 25 2627 28 Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- 29 30 31 32 33 34 3536 37 Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly where the Gly at position 37is substituted by a Lys and the Ala at position 8 is substituted by Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid or(1-aminocyclooctyl) carboxylic acid and where said GLP-1 analog isacylated with a diacid attached directly to the Lys at position
 37. 2.The acylated GLP-1 analog of claim 1, wherein the Lys at position 26 andthe Lys at position 34 of SEQ ID NO: 1 are each substituted by an Arg.3. The acylated GLP-1 analog of claim 2, wherein the His at position 7has been substituted by imidazopropionyl, α-hydroxy-histidine,α-hydroxy-desamino-histidine, N-methyl-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, α-fluoromethyl-histidine,α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or4-pyridylalanine.
 4. A pharmaceutical composition comprising an acylatedGLP-1 analog according to claim 1 and a pharmaceutically acceptableexcipient.
 5. An acylated GLP-1 (7-37) analog where said GLP-1 analoghas a sequence according to SEQ ID NO: 1 where the Gly at position 37 issubstituted by a Lys, the His at position 7 is substituted byimidazopropionyl, α-hydroxy-histidine, α-hydroxy-desamino-histidine,N-methyl-histidine, D-histidine, desamino-histidine, 2-amino-histidine,β-hydroxy-histidine, homohistidine, N^(α)-acetyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine or 4-pyridylalanine, the Ala at position 8 issubstituted by Gly, Val, Ile, Leu, Ser or Thr, and where said GLP-1analog is acylated with a diacid attached directly to the Lys atposition
 37. 6. The acylated GLP-1 analog of claim 5, wherein the Lys atposition 26 and the Lys at position 34 are each substituted by an Arg.7. The acylated GLP-1 analog of claim 6, wherein the Ala at position 8is substituted by Ser.
 8. A pharmaceutical composition comprising anacylated GLP-1 analog according to claim 5 and a pharmaceuticallyacceptable excipient.